Insulin's effect on protein phosphorylation is complex. When given with hormones that raise levels of c-AMP, insulin inhibits c-AMP dependent protein phosphorylation. When given as the sole hormone, however, insulin has been shown by the principal investigator and others to stimulate the phosphorylation of a distinct set of adipocyte or hepatocyte proteins, and without a change in total c-AMP levels. This project investigates the effect of insulin on protein phosphorylation in a third target cell, muscle. Cultured I skeletal muscle cells are incubated with 32Pi until steady-state formation of 326P-phosphopeptides, and then exposed to hormone for 20 minutes. Following sonication and polyacrylamide gel electrophoresis, autoradiographs are made from dried gels and 32Pi incorporation is quantitated by densitometry. Initial studies in this project have demonstrated that insulin stimulates the phosphorylation of one cellular peptide, that of MW 29,000 daltons. This effect is maximum by 20 minutes, and can be seen with 500 U/ml insulin, a concentration similar to that required for insulin stimulation of glycogen synthesis in these cells. The 29,000 MW peptide is located in the 105,000g pellet, a site consistent with placement in the plasma membrane or cellular microsomes. The proposed studies will attempt to categorize this insulin effect as a "growth" effect, often seen incultured cells, or a "metabolic" effect, seen in physiologic target tissues. Furthermore, the effect of insulin will be contrasted to that of agents which increase c-AMP levels, and the competitive effect of insulin described above looked for. Studies will also proceed to isolate the insulin-responsive phosphopeptide and establish the functional significance of this change.